Reagent composition and process for determining gamma-glutamyltranspeptidase

ABSTRACT

A composition comprising gamma -glutamyl-p-nitroaniline, glucyl-glycine, a buffer, a surface-active agent and polyvinylpyrrolidone is an exceptionally stable reagent for determining gamma -glutamyl-transpeptidase.

United States Patent [191 Bernt et a1.

[73] Assignee: Boehringer Mannheim GmbH,

Mannheim, Germany [22] Filed: Aug. 11, 1971 [21] Appl. No.: 170,968

[30] Foreign Application Priority Data Aug. 28, 1970 Germany P 20 42 829.9

[52] U.S. Cl. 195/l03.5 R, 195/99 Nov. 20, 1973 [51] Int. Cl. G01n 31/14 [58] Field of Search 195/103.5 R, 99

[56] References Cited UNITED STATES PATENTS 11/1972 Nakanishi et al. l95/l03.5 R X 7/1970 Kita 23/230 B X Primary ExaminerLionel M. Shapiro Assistant Examiner-Max D. Hensley Attorney-Ralph D. Dinklage et al.

[57] ABSTRACT A composition comprising -glutamyl-p-nitroaniline, glucyl-glycine, a buffer, a surface-active agent and polyvinyl-pyrrolidone is an exceptionally stable reagent for determining 'y-glutamyl-transpeptidase.

11 Claims, No Drawings 1 REAGENT COMPOSITION AND PROCESS FOR DETERMINING GAMMA-GLUTAMYLTRANSPEPTIDASE presence of a suitable buffer.

To an increasing extent, the determination of 'y-glutarnyl-transpeptidase (y-GT) is carried out in the clinical chemical laboratory for the diagnosis of liver diseases. This determination takes place, via a known reaction, according to the equation: 'y-glutamyl-p-nitroaniline glycyl-glycine nitroaniline glutamyl-(glycyl-glycine) The velocity of liberation of the yellow-colored pnitroaniline can be followed optically and is a measure of the activity of 'y-GT present.

For a routine use of this process, the instability and poor solubility of the substrate 'y-glutamyl-pnitroaniline is a severe disadvantage. This compound can only be brought into' solution with difficulty and solutions thereof are only stable for a very short time. Therefore, it was previously necessary to prepare a small amount of solution sufficient only for relatively few determinations, which had to be carried out within a short period of time. It was not possible to store the reagent solution.

The present invention substantially overcomes this drawback of the prior art compositions by providing a reagent which does not exhibit the above described instability.

The present invention is based upon the surprising discovery that the use of polyvinyl-pyrrolidone, together with a surface-active agent, substantially and decisively improves the solubility properties and the stability of 'y-glutamyl-p-nitroaniline.

Thus, according to the present invention, there is provided a reagent composition for the determination of 'y-glutamyl-transpeptidase which comprises -y-glutamyl-p-nitroaniline, glycyl-glycine and a buffer, together with polyvinyl-pyrrolidone and a surface-active agent.

The reagent according to the present invention preferably also contains an azide, especially an alkali metal azide, for example sodium azide.

As surface-active agent, the reagent according to the present invention can contain any such agent which does not exert an inhibiting or inactivating influence on the -y-glutamyl-transpeptidase. The hydroxypolyethoxyalkanes, especially hydroxypolyethoxydodecane, have proved to be especially useful.

The agent according to the present invention preferably contains the 'y-glutamyl-p-nitroaniline in lyophilized form, optionally together with the other components of the reagent in solid form. More preferably, however, the reagent according to the present invention consists of two mixtures, one of which contains 'y-glutamyl-p-nitroaniline and glycyl-glycine in lyophilized form and the other consists of an aqueous solution, which contains the buffer, polyvinyl-pyrrolidone, the surface-active agent and optionally an azide. Both of the mixtures are stable for a long time and, by simple mixing, with gentle warming, permit the rapid preparation of a relatively stable solution, which remains usable for a comparatively long time.

In a preferred specific embodiment, the aqueous solution comprises:

0.05 to 0.2 M tris (tris-(hydroxyalkylamine)) buffer,

pH 7.8 to 8.2,

20 to 100 grams/liter polyvinyl-pyrrolidone,

0.75 to 5 grams/liter surface-active agent and optionally 0.5 to 4 grams/liter of an azide.

in an especially preferred specific embodiment, the aqueous solution comprises:

0.1 to 0.2 M tris buffer, pH 8.0,

30 grams/liter polyvinyl-pyrrolidojne,

l gram/liter hydroxypolyethoxyalkane and l gram/liter sodium azide.

The second component of the reagent according to the present invention, i.e., the solid mixture of y-glutamyl-p-nitroaniline and glycyl-glycine, preferably comprises:

1.0 to 1.25 grams, preferably 1.13 grams,

y-glutamyl-p-nitroaniline and 5.0 to 6.2 grams, preferably 5.6 grams, glycylglycine, per liter of liquid reagent component.

The reagent according to the present invention permits the preparation of solutions ready to use for the determination of y-glutamyl-transpeptidase, which solutions remain usable for a comparatively long period of time, in contradistinction to the previously known solutions of 'y-glutamyl-p-nitroaniline which, on the one hand, were difficult to prepare and from which, on the other hand, this compound crystallized out again a short time after preparation of the solution. Thus, for the first time, the reagent according to the present invention provides the possibility of carrying out the routine determination of 'y-glutamyl-transpeptidase, for example, in automatic analysis devices.

The following Examples are given for the purpose of illustrating the present invention and should not be construed as unduly limitative thereof.

PREPARATION OF THE REAGENT a. Lyophilized substrate 6.85 grams y-glutamyl-p-nitroaniline and 33.8 grams glycyl-glycine. are dissolved, with warming to 70 to 80C., in 6 liters of double distilled water. The warm solution was tilled in 3.0 ml. portions into 200 small bottles, frozen therein and lyophilized in the usual way. The preparations so obtained were stable at ambient temperature for at least 12 months.

b. Preparation of the buffer solution 193.5 grams tris-(hydroxymethylamine)-methane, 240 grams polyvinyl-pyrrolidone, 8 grams polyethoxydodecane (commercially available under the trade name Thesit) and 8 grams sodium azide were dissolved in 8 liters of double distilled water. The solution is adjusted to pH 8.0 with dilute hydrochloric acid. The solution thus obtained was of unlimited stability at ambient temperature. c. Activity determination of 'y-glutamyl-transpeptidase The lyophilized prepared was described under (a) above was suspended in 3 m1. of the solution prepared according to (b) above and, by brief warming to about was followed, the velocity of which was a measure of the 7-glutamyl-transpeptidase activity.

d. Influence of polyvinyl-pyrrolidone (PVP) and hydroxypolyethoxydodecane (HPD) on the solubility of 'y-glutamyl-p-nitroaniline l 1.4 grams -y-glutamyl-p-nitroaniline and 56.4 grams glycyl-glycine were dissolved, with warming to 70 to 80C., in 10 liters double distilled water. The warm solution was filled, in 3.0 ml. portions, into small bottles, frozen in an lyophilized.

Solubility experiments were then carried out with the lyophilizates thus obtained. The lyophilizates were dissolved at 50 to 70C. in 0.2M tris buffer which contained (a) no additive and (b) PVP and HPD in a definite concentration ratio, the solution cooled to 25C. and the solubilizing properties of PVP and HPD determined either by measurement of the turbidity (percent light permeability) or by observation of the crystallization time. The results obtained are set out in the following Table:

% PVP 3 HPD O I initially 85% 84% after 3 hours 60% 85% after 6 hours 50% 84% after 24 hours 20% 84% after 2 days crystallizes 82% after 6 days crystallizes 78% It will be understood that the specification and examples are illustrative but not limitative of the present invention and that other embodiments within the spirit and scope of the invention will suggest themselves to those skilled in the art.

What is claimed is:

l. Reagent composition for the determination of 'y-glutamyl-transpeptidase which composition comprises y-glutamyl-p-nitroaniline, glycyl-glycine, a tris buffer, a hydroxypolyethoxyalkane surface-active agent and polyvinylpyrrolidone.

2. Composition as claimed in claim 1 additionally containing an azide.

3. Composition as claimed in claim 2 wherein said azide is an alkali metal azide.

4. Composition as claimed in claim 1 wherein the surface-active agent is an hydroxypolyethoxydodecane.

5. Composition as claimed in claim 1 wherein the buffer is a tris -(hydroxyalkylamine).

6. Composition as claimed in claim 1 comprising:

A. 0.05 to 0.2M tris buffer, pH 7.8 to 8.2,

20 to I00 grams polyvinyl-pyrrolidone, 0.75 to 5 grams of a surface-active agent, 0.5 to 4 grams of an azide, and

B. a lyophilized mixture of,

1 to 1.25 grams 'y-glutamyl-p-nitroaniline and 5 to 6.2 grams glycyl-glycine per liter of buffer solution.

7. Composition as claimed in claim 1 comprising:

A. 0.1 to 0.2M tris buffer, pH 8.0,

30 grams polyvinyl-pyrrolidone,

1 gram hydroxypolyethoxyalkane, 1 gram sodium azide, and

B. 1.13 grams 'yglutarnyl-p-nitroaniline and 5.6 grams glycyl-glycine. per liter of buffer solution.

8. Composition as claimed in claim 6 wherein the azide is sodium azide.

9. Method for determining 'y-glutamyl-transpeptidase which comprises contacting a test sample suspected of containing y-glutamyl-transpeptidase with a diagnostic reagent composition comprising 'y-glutamyl-pnitroaniline, glycyl-glycine, a tris buffer, a hydroxypolyethoxyalkane surface-agent and polyvinylpyrrolidone, and following the liberation of pnitroaniline as a measure of any activity of 'y-glutamyltranspeptidase present in the test sample.

10. Method as claimed in claim 9 which comprises forming a reagent composition by contacting, per liter of solution,

A. 0.05 to 0.2M tris buffer, pH 7.8 to 8.2,

20 to grams polyvinyl-pyrrolidone, 0.75 to 5 grams of a surface-active agent, 0.5 to 4 grams of an azide in the form of an aqueous solution with B. a lyophilized mixture of buffer 1 to 1.25 grams 'y-glutamyl-p-nitroaniline and 5 to 6.2 grams glycyl-glycine; per liter of solution, and then contacting a test sample with the resulting mixture and observing the liberation of yellow-colored y-glutamyl-p-nitroaniline.

11. Method as claimed in claim 10 which comprises forming a reagent composition by contacting, per liter of solution,

A. 0.1 to 0.2M tris buffer, ph 8.0

30 grams polyvinyl-pyrrolidone, 1 gram hydroxypolyethoxyalkane, 1 gram sodium azide, and in the form of an aqueous solution with B. l.l3 grams 'y-glutamyl-p-nitroaniline and 5.6 grams glycyl-glycine; per liter of solution, and then contacting a test sample with the resulting mixture and observing the liberation of yellow-colored 'y-glutamyl-p-nitroaniline.

* t I? k 

2. Composition as claimed in claim 1 additionally containing an azide.
 3. Composition as claimed in claim 2 wherein said azide is an alkali metal azide.
 4. Composition as claimed in claim 1 wherein the surface-active agent is an hydroxypolyethoxydodecane.
 5. Composition as claimed in claim 1 wherein the buffer is a tris -(hydroxyalkylamine).
 6. Composition as claimed in claim 1 comprising: A. 0.05 to 0.2M tris buffer, pH 7.8 to 8.2, 20 to 100 grams polyvinyl-pyrrolidone, 0.75 to 5 grams of a surface-active agent, 0.5 to 4 grams of an azide, and B. a lyophilized mixture of, 1 to 1.25 grams gamma -glutamyl-p-nitroaniline and 5 to 6.2 grams glycyl-glycine per liter of buffer solution.
 7. Composition as claimed in claim 1 comprising: A. 0.1 to 0.2M tris buffer, pH 8.0, 30 grams polyvinyl-pyrrolidone, 1 gram hydroxypolyethoxyalkane, 1 gram sodium azide, and B. 1.13 grams gamma -glutamyl-p-nitroaniline and 5.6 grams glycyl-glycine, per liter of buffer solution.
 8. Composition as claimed in claim 6 wherein the azide is sodium azide.
 9. Method for determining gamma -glutamyl-transpeptidase which comprises contacting a test sample suspected of containing gamma -glutamyl-transpeptidase with a diagnostic reagent composition comprising gamma -glutamyl-p-nitroaniline, glycyl-glycine, a tris buffer, a hydroxypolyethoxyalkane surface-agent and polyvinyl-pyrrolidone, and following the liberation of p-nitroaniline as a measure of any activity of gamma -glutamyl-transpeptidase present in the test sample.
 10. Method as claimed in claim 9 which comprises forming a reagent composition by contacting, per liter of solution, A. 0.05 to 0.2M tris buffer, pH 7.8 to 8.2, 20 to 100 grams polyvinyl-pyrrolidone, 0.75 to 5 grams of a surface-active agent, 0.5 to 4 grams of an azide in the form of an aqueous solution with B. a lyophilized mixture of buffer 1 to 1.25 grams gamma -glutamyl-p-nitroaniline and 5 to 6.2 grams glycyl-glycine; per liter of solution, and then contacting a test sample with the resulting mixture and observing the liberation of yellow-colored gamma -glutamyl-p-nitroaniline.
 11. Method as claimed in claim 10 which comprises forming a reagent composition by conTacting, per liter of solution, A. 0.1 to 0.2M tris buffer, ph 8.0 30 grams polyvinyl-pyrrolidone, 1 gram hydroxypolyethoxyalkane, 1 gram sodium azide, and in the form of an aqueous solution with B. 1.13 grams gamma -glutamyl-p-nitroaniline and 5.6 grams glycyl-glycine; per liter of solution, and then contacting a test sample with the resulting mixture and observing the liberation of yellow-colored gamma -glutamyl-p-nitroaniline. 